The present invention relates to a process for producing a physiologically active polypeptide, more particularly, to a process for producing active human IL-18.
IL-18, also known as interferon-γ-inducing factor, is a recently discovered novel cytokine. Active IL-18 contains 157 amino acid residues. It has potent biological activities, including induction of interferon-γ-production by T cells and splenocytes, enhancement of the killing activity of NK cells and promotion of the differentiation of naïve CD4+T cells into Th1 cells. In addition, human IL-18 augments the production of GM-CSF and decreases the production of IL-10. IL-18 has been shown to have greater interferon-γ inducing capabilities than IL-12 and signals through a different receptor and utilizes a distinct signal transduction pathway.
IL-18, the encoding nucleotide sequence, and certain physicochemical chemical properties of the purified protein is known (Ushio, S., et aL, 1996, J. Immunology, 156, 4274–4279; Dinarello, C. A., et al. 1998, J. Leukocyte Biology, 1998. 63, 658–664).
Kabushiki Kaisha Hayashibara Seibutsu Kayaku Kenkyujo's (“Hayashibara”), U.S. Pat. No. 5,192,324, which corresponds to EP 0692536, published on Jan. 17, 1996, discloses a mouse protein which induces IFN-gamma production by immunocompetent cells, the protein being further characterized as having certain physicochemical properties and a defined partial amino acid sequence. Also disclosed is a protein having a 157 aa sequence, two fragments thereof, DNA (471 bp) encoding the protein, hybridomas, protein purification methods, and methods for detecting the protein.
Hayashibara's U.S. Pat. No. 6,214,584, which corresponds to EP 0712931, published on May 22, 1996, discloses a 157 aa human protein and homologues thereof, DNA encoding the protein, transformants, processes for preparing the protein, monoclonal antibodies against the protein, hybridomas, protein purification methods, methods for detecting the protein, and methods of treatment and/or prevention of malignant tumors, viral diseases, bacterial infectious diseases, and immune diseases.
Incyte Pharmaceuticals, Inc.'s, WO 97/24441, published on Jul. 10, 1997, discloses a 193 amino acid protein corresponding to IL-18 precursor and encoding DNA.
In human cells, polypeptides formed by the expression of genes may be processed by intracellular enzymes to be partially digested and to receive sugar chains. Polypeptides to be satisfactorily incorporated into pharmaceuticals may be those which were processed similarly as in human cells. It is known that most cytokines are usually produced as precursors with no biological activity, and then processed by intracellular enzymes to be converted into active polypeptides.
The IL-18 polypeptide usually exists in human cells in the form of a precursor of 193 amino acids and no biological activity. The precursor IL-18 is also referred to as Pro-IL-18. One method of producing active IL-18 from its precursor is taught by Hayashibara's U.S. Pat. No. 5,879,942, which corresponds to EP 0819757, published on Jan. 21, 1998. The patent discloses an enzyme or a protein which converts a precursor of IL-18 into active IL-18.
Another method of producing active IL-18 from its precursor is taught by Hayashibara's U.S. Pat. No. 5,891,663, which corresponds to EP 0821005, published on Jan. 28, 1998. The patent discloses contacting precursor IL-18 with interleukin-1β-converting enzyme (“ICE”). The teachings of the patents and references are incorporated by reference.
The role of ICE as a mediator of apoptosis and inflammation has been extensively studied in the literature. It is also known that ICE can process precursors of both Interleukin-1 and Interleukin-18 to active forms (Thomberry, N A, et al., 1992, Nature 356, 768–774; Gbayur, T et al., 1997, Nature 386, 619–623).